Protein therapeutics that blocks the activation of IgE hold the promise of being used as the next generation of drugs in the treatment of asthma. Indeed, omalizumab, a humanized monoclonal antibody that inhibits the binding of IgE-Fc to the high-affinity IgE receptor Fc5RI, has beneficial effects in the treatment of moderate to severe allergic asthma in patients whose symptoms are inadequately controlled. Despite its effectiveness, omalizumab treatment is only limited in patients with very severe disease, presumably due to the very high cost of this monoclonal antibody drug. This project is directed at developing IgE-Fc-binding single domain antibodies (SDAs) for cost-effective treatment of asthma. Specifically, we will use a directed protein evolution method called mRNA-display to develop a new generation of IgE-Fc-binding SDAs that are based on a newly identified monomeric human VH domain (hVH). We will first generate SDAs that tightly and specifically bind to the Fc5RI-binding region on human IgE-Fc from an mRNA-displayed hVH domain library containing more than 1013 unique sequences through amplification-based, iterative rounds of in vitro selection. The selected sequences will be optimized to acquire high target-binding affinity and specificity through a combination of special procedures. The resulting SDAs will be characterized in detail for their target-binding properties using various in vitro and in vivo approaches. The optimized SDA will be dimerized to increase its binding strength with IgE-Fc. The resulting IgE-Fc-binding SDAs based on hVH domain would have a number of advantages, including high target-binding affinity and specificity, low immunogenicity, and significantly reduced manufacturing costs due to their high expression levels in bacteria. Such SDAs could have significant applications in the treatment of asthma. PUBLIC HEALTH RELEVANCE: Asthma is a major global health problem with an enormous increase in prevalence during the past few decades. In the United States alone, about 20 million people, including nearly 9 million children, have asthma. Despite the availability of effective and relatively cheap treatments that benefit the majority of asthma patients, approximately 5% of severe asthmatic patients remain poorly controlled. The proposed research aims at developing a novel class of anti-asthma human single domain antibodies that possess unique features, including high efficacy, low immunogenicity, and significantly reduced manufacturing cost. The availability of such biopharmaceuticals could greatly benefit many patients with severe-symptomatic asthma.